Arboriculture & Urban Forestry 37(6): November 2011 MATERIALS AND METHODS Experimental Design and Treatments The study was conducted in 48 individual, physically isolated soil cells (phytotrons) located at the Ohio Agricultural Research and Development Center in Wooster, Ohio, U.S. Each phytotron was constructed by slicing a narrow trench one meter deep, around plots 4 m2 in area. The perimeter of each phytotron was lined with 30 mil PVC landfill liner extending one meter vertically into and 20 cm above the ground, which was heat welded at the overlap to completely enclose the plot. The liner isolated the soil environment without affecting drainage, allowing for establishment of repli- cated, randomized soil environments. The native soil is a Canfield silt loam (29% sand, 42% silt, 29% clay). A single paper birch (avg. height: 1.1 ± 0.07 m) was planted in the corner of each plot on September 20, 2000, and a single Austrian pine (avg. height: 1.0 ± 0.12 m) was added to the opposing corner of each plot on September 12, 2002. Weeds were controlled with pre-emergence herbicides (isoxaben and oryzalin or prodiamine) in combina- tion with manual cultivation and spot treatment with glyphosate. The experiment was designed as a randomized complete block, with four treatments: 1) untreated control, 2) paclobutra- zol soil drench, 3) fertilizer, and 4) paclobutrazol + fertilizer. There were 12 replicate trees per treatment, with each treatment combination replicated four times in each of three blocks. Pa- clobutrazol was applied via soil drench on September 19, 2002, to both paper birch and Austrian pine using the commercial prod- uct Cambistat 2SC (22.3% paclobutrazol) at the labeled rate of 17 ml per 2.54 cm trunk diameter. Trunk diameter was measured at 50 cm from ground level. The fertilization treatment consisted of 3:1:1 N:P:K with 60% of N in slow release form (urea form- aldehyde) and 40% in fast release form (ammonium and nitrate) applied to the soil surface at a rate of 200 Kg N/ha/yr, with half the annual rate applied just after bud break in the spring, and the other half prior to leaf drop in late summer or early autumn. The fertilization treatment represents an intermediate rate based on standard recommendations for woody plants (ANSI 2004). Trees were fertilized in May and September each year through 2005, with fertilization of paper birch and Austrian pine com- mencing in May 2001 and September 2002, respectively. Treatment effects on tree physiology, secondary metabo- lism, and insect resistance were measured in 2003 and 2004 for paper birch, and from 2003 to 2005 for Austrian pine. Pine trees were maintained for an additional year to test for any lagging effects of treatments on resistance to European pine sawfly, which feeds on needles that are one year old or older. Tree Growth, Photosynthesis, and Leaf Morphology Trunk diameter, measured 50 cm aboveground, and tree height were measured at planting and in late autumn from 2003 to 2004 for both paper birch and Austrian pine, and again in late autumn 2005 for Austrian pine. Annual tree growth was calculated by subtracting consecutive measurements for diameter and height. Light-saturated photosynthesis was quantified using a LI- COR LI-6200 Portable Photosynthesis System (LI-COR, Inc., Lincoln, Nebraska, U.S.). Measurements were taken from pa- per birch on June 25, July 25, and August 14 in 2003, and July 21 in 2004; and from Austrian pine on August 24, 2004. Leaves sampled were standardized for exposure and age, and mea- 279 surements were made during cloud-free mornings (9:00–11:00 am) to ensure that measurements reflected treatment effect on tree physiology rather than other environmental factors af- fecting stomatal aperture. The blocking design of the experi- ment provided additional control over environmental variation. Leaf area and specific leaf mass were quantified for paper birch on June 24, 2003 and July 7, 2004 by sampling 12 leaves throughout the canopy. Leaf age and exposure were standardized by sampling the youngest fully mature leaves with full expo- sure to sunlight. Austrian pine foliage was sampled on October 24, 2004 and June 29, 2005 by collecting from throughout the canopy 12 needle fascicles that had formed in the previous year. Leaf area of 12 sampled birch leaves per tree was determined using imaging software [CI-400 Computer Image Analysis Sys- tem and software (CID, Inc.)], and for each pine tree by mea- suring the length and diameter of one needle from each fascicle (Svenson and Davies 1992), after which leaves/needles were dried to constant weight (85°C for 72 hours). Specific leaf and needle mass (g/m2 ) was determined for birch and pine, respec- tively, by dividing sampled leaf/needle dry mass by its area. centrations were quantified using the Folin-Denis assay. These colorimetric methods were modified for use with an automated continuous flow analyzer as documented in detail by Nitao et al. (2001). Commercially available “ground ordinary quebra- cho tannin” (L.H. Lincoln & Son, Inc., Coudersport, Pennsyl- vania, U.S.) was used as an internal standard. Calibration curves for the internal standard in relation to purified tannins (either from red pine or paper birch foliage) were used to quantify tannins and phenolics in the samples, with concentrations re- ported as tannic acid equivalents on a percent dry mass basis. Foliar terpene concentrations of one-year-old Austrian pine needles were quantified on three dates (May 9, 2003; May 6, 2004; and May 10, 2005). Immediately upon sampling, needles were flash-frozen in the field in liquid nitrogen, placed in a cooler on ice, and then transported to the lab, where they were stored at -80°C until analysis. Concentrations of monoterpenes (α-pinene, β-pinene, myrcene, camphene, limonene, and bornyl-acetate), and sesquiterpenes (trans-caryophyllene, alpha-humulene, and germacrene-D) were analyzed via gas chromatography (GC- FID) following extraction of 1 g of fresh weight needles in 10 Phytochemistry Nitrogen concentration was quantified for paper birch fo- liage once in 2003 (June 24) and twice in 2004 (June 14 and August 24), and for Austrian pine previous-year needles on May 6, 2004 and May 10, 2005. Leaf and needle samples were freeze-dried at -4°C using an FTS temperature-controlled tray freezedryer, and ground in a mill (Cyclotec EC 1093, Teca- tor AB, Hoganas, Sweden) to pass through a 0.4 mm mesh screen. A subsample of 7 mg of this fine powder was used to quantify total leaf and needle nitrogen using a Carbo Erba CHN analyzer, Model NA 1500 (Daun and DeClerq 1994). Foliar concentrations of condensed tannins and total phenolic compounds were quantified on three dates for paper birch (June 24, 2003; June 14, 2004; and August 24, 2004), and for Aus- trian pine with previous-year foliage sampled on May 9, 2003 and May 6, 2004, and current-year foliage sampled on August 24, 2004. Condensed tannins (proanthocyanidins) were quanti- fied using the H2 SO4 method, and total foliar phenolics con- ©2011 International Society of Arboriculture
November 2011
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